Compositions and methods for treatment of anticancer-drug resistant cancers
Embodiments of the instant disclosure relate to novel methods and compositions for treating tumors resistant to one or more anticancer drugs, such as platinum-based chemotherapeutics.
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This application is a continuation of PCT Application No. PCT/US2021/026164, filed on Apr. 7, 2021, which claims the benefit of Chinese Patent Application No. 202010271412.4, filed on Apr. 7, 2020, each of which is hereby incorporated by reference in its entirety.
GOVERNMENTAL RIGHTSThis invention was made with government support under CA184717 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELDThe present disclosure generally relates to compositions and methods for the treatment of cancer and tumor cells, prevention of tumor cell metastasis, and treatment and prevention of cancers and of cancers resistant to one or more anticancer drugs, such as platinum-based chemotherapeutics.
BACKGROUNDAnticancer drugs, such as platinum-based chemotherapy regimens, are frequently a standard of care for cancer patients, particularly those with late stage cancer and metastatic disease. A cancer that responds to a first anticancer drug treatment only to have the cancer return within a certain period is considered an anticancer drug-resistant cancer. For those with anticancer drug-resistant cancers, positive outcomes are very likely be low.
Several anticancer drugs work by producing excessive DNA damage that causes cell death directly or following DNA replication. However, DNA repair pathways can repair lesions induced by anticancer drugs, enabling cancerous tumor cells to survive and ultimately become resistant to the DNA damage that is induced by anticancer drug treatments. As such, there is a need in the art for cancer therapies that target the DNA repair pathways of cancerous cells. Once identified, cancer therapies capable of inhibiting DNA repair could be administered in combination with anticancer drugs to increase efficacy of their DNA-damaging effects in cancerous cells and tumors.
SUMMARYEmbodiments of the instant disclosure relate to novel methods and unique compositions for treating cancers, tumors (e.g., solid tumors), tumors resistant to anticancer drugs (e.g., platinum-based chemotherapy) and/or tumors suspected of developing anticancer drug resistance in a subject.
Certain embodiments of the present disclosure provide for compositions for treating cancer. In some embodiments, compositions for treating one or more cancers described herein can include at least one compound represented by formula I or a pharmaceutically acceptable salt thereof:
wherein, R1 is independently mono- or poly-substituted with hydrogen, trifluoromethyl, trifluoromethoxy, nitro, cyano, halogen, C1-C10 alkyl, or —OR2, wherein R2 is independently selected from hydrogen or C1-C10 alkyl.
In some embodiments, compositions herein may have at least one compound represented by formula I or a pharmaceutically acceptable salt thereof wherein the compound of formula I may be selected from one or more compounds selected from the group consisting of:
or any combination thereof. In some embodiments, compositions herein may comprise at least one compound represented by formula III.
In some embodiments, compositions for treating cancers as disclosed herein can further comprise one or more anticancer drugs. In some embodiments, compositions for treating cancers as disclosed herein can further comprise one or more platinum-based chemotherapeutics. In accordance with some embodiments herein, the one or more platinum-based chemotherapeutics may comprise one or more of cisplatin, carboplatin, nedaplatin, satraplatin, picoplatin, phenanthriplatin, triplatin tetranitrate, or any combination thereof. In some embodiments, compositions for treating cancers as disclosed herein can further comprise gemcitabine, methotrexate, vinblastine, adriamycin, or any combination thereof. In some embodiments, compositions for treating cancers as disclosed herein may further comprise one or more pharmaceutically acceptable excipients.
Certain embodiments of the present disclosure provides for compounds represented by formula I or a pharmaceutically acceptable salt thereof. In some embodiments, compounds of the present disclosure represented by formula I or a pharmaceutically acceptable salt thereof may be a compound selected from the group consisting of any one of the compounds represented by formulas II-XVI or any one of their pharmaceutically acceptable salts. In some embodiments, compounds of the present disclosure represented by formula I or a pharmaceutically acceptable salt thereof may be a compound represented by formula III. In some embodiments, a compound for use in the compostions and methods of the present disclosure may be (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol.
In some embodiments, the present disclosure provides for pharmaceutical compositions comprising any one or more of the compounds or compositions disclosed herein. In some embodiments, pharmaceutical compositions disclosed herein may further include at least one pharmaceutically acceptable excipient.
Certain embodiments of the present disclosure provide for methods of delivering at least one of the compositions, compounds, and/or pharmaceutical compositions disclosed herein to a cell. Methods herein can include introducing the cell to any one of the compositions, compounds, and/or pharmaceutical compositions disclosed herein. In accordance with some embodiments herein, a cell to be introduced to any one of the compositions, compounds, and/or pharmaceutical compositions disclosed herein may be a cancer cell, a tumor cell, or any combination thereof.
Certain embodiments of the present disclosure provide for methods of delivering at least one of the compositions, compounds, and/or pharmaceutical compositions disclosed herein to a tumor. Methods herein can include introducing the tumor to any one of the compositions, compounds, and/or pharmaceutical compositions disclosed herein. In accordance with some embodiments herein, a tumor to be introduced to any one of the compositions, compounds, and/or pharmaceutical compositions disclosed herein may be a testicular tumor, ovarian tumor, cervical tumor, kidney tumor, bladder tumor, head-and-neck tumor, liver tumor, stomach tumor, lung tumor, endometrial tumor, esophageal tumor, breast tumor, cervical tumor, central nervous system tumor, germ cell tumor, prostate tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, neuroblastoma, sarcoma, multiple myeloma, melanoma, mesothelioma, osteogenic sarcoma, or any combination thereof. In some examples, a tumor to be introduced to any one of the compositions, compounds, and/or pharmaceutical compositions disclosed herein may be a breast tumor, an ovarian tumor, or any combination thereof.
Certain embodiments of the present disclosure provide for methods of treating a tumor in a subject in need thereof. In some embodiments, methods disclosed herein for treating a tumor in a subject in need thereof may comprise administering to the subject at least one of the compositions, compounds, and/or pharmaceutical compositions disclosed herein.
In accordance with some embodiments herein, a subject in need thereof can be a subject undergoing or will undergo at least one anticancer drug therapy. In some embodiments, a subject in need thereof can be a subject undergoing or will undergo at least one or more platinum-based chemotherapeutics. In some embodiments, a subject may be in need of treating a tumor using the methods disclosed herein if the subject has a progressive cancer through the at least one anticancer drug therapy, or is resistant to the at least one anticancer drug therapy.
Certain embodiments of the present disclosure provide for methods of preparing the compounds disclosed herein. In some embodiments, methods of preparing at least one compound disclosed herein can have one or more of the following steps: (i) reacting a compound represented by Compound A with concentrated hydrochloric acid and paraformaldehyde to obtain a compound represented by Compound B; (ii) reacting a compound represented by Compound B with triphenylphosphonium in an aprotic solvent to obtain a compound represented by Compound C; (iii) producing a compound represented by Compound E from compounds represented by Compound C and Compound D under strong base conditions; (iv) reacting a compound represented by Compound E with trifluoromethanesulfonic anhydride in dichloromethane to obtain a compound represented by Compound F; (v) reacting a compound represented by Compound F with pinacol diboronic acid or diboronic acid under palladium salt catalyst to obtain a compound represented by Compound G; and (vi) reacting a compound represented by Compound G with a reducing agent such and adding acid to obtain a compound represented by Compound H, wherein the reducing agent comprises sodium borohydride, tetrahydroaluminum lithium, or any combination thereof;
wherein:
wherein: R1 is independently mono- or poly-substituted with hydrogen, trifluoromethyl, trifluoromethoxy, nitro, cyano, halogen, C1-C10 alkyl, or —OR2, wherein R2 is independently selected from hydrogen or C1-C10 alkyl.
In some embodiments, methods of preparing at least one compound disclosed herein can have strong base conditions of step (iii) that can be strong alkaline conditions comprising sodium methoxide, sodium ethoxide, sodium tert-butoxide, sodium isopropoxide, butyl lithium, sodium hydroxide, lithium hydroxide, potassium hydroxide, or any combination thereof. In some embodiments, methods of preparing at least one compound disclosed herein can have steps (iii) and (v) performed at about 80° C. In some embodiments, methods of preparing at least one compound disclosed herein can have steps (iv) and (vi) performed at about 0° C.
In some embodiments, methods of preparing at least one compound disclosed herein can comprise palladium salt catalyst in step (v). In some examples, the palladium salt catalyst may be palladium acetate, palladium chloride, tetrakis(triphenylphosphorus) palladium, bis(acetonitrile) palladium dichloride, bis(triphenylphosphorus) palladium chloride, 1,1′-[bis(diphenylphosphorus)ferrocene]palladium dichloride, bis(benzonitrile)palladium dichloride, 1,1′-[bis(di-tert-butylphosphorus)ferrocene] Palladium dichloride, bis(tricyclohexylphosphorus) palladium dichloride, bis(o-toluene) palladium dichloride, or any combination thereof.
In some embodiments, methods of preparing at least one compound disclosed herein may comprise step (v) performed under an alkaline condition, wherein the alkaline condition may be a base comprising sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, sodium fluoride, potassium fluoride, or any combination thereof.
In some embodiments, methods of preparing at least one compound disclosed herein may comprise step (v) performed in a polar solvent, wherein the polar solvent may be N, N-dimethylformamide, dimethylsulfoxide, acetonitrile, acetone, methyl ethyl ketone, 1,4-dioxane, water, or any combination thereof.
In certain embodiments, kits are provided herein. In certain embodiments, compositions of the instant disclosure can be included in the kit, together or in separate containers. In certain embodiments, kits are provided for the practice of any one of the methods disclosed herein. In some embodiments, kits can include one or more of the compounds disclosed herein, one or more anticancer drugs (e.g., platinum-based chemotherapeutics), or a combination thereof.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office by request and payment of the necessary fee. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.
In the following sections, certain exemplary compositions and methods are described in order to detail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details can be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.
Embodiments of the instant disclosure relate to novel methods and compositions for treating tumors resistant to or suspected of becoming resistant to one or more anticancer drugs, such as platinum-based chemotherapeutics. In some embodiments, a tumor subjected to the methods and compositions disclosed herein can be a solid tumor. In some embodiments, compositions and methods disclosed herein are designed to re-sensitize or sensitize a tumor in a subject to an anticancer drug to reduce costs, improve outcome, and reduce or eliminate patient exposure to anticancer therapy without significant effect.
I. Resveratrol Derivatives and And-1 Inhibitors
And-1, also known as WD repeat and HMG-box DNA binding protein 1 (WDHD1) and chromosome transmission fidelity factor 4 homolog (Ctf4), is an acidic nucleoplasmic DNA-binding protein containing N-terminal WD40 domains, a middle SepB domain, and a C-terminal high mobility group (HMG) domain. And-1 is important in regulating DNA replication, chromosome function, and DNA damage repair; however, a potent And-1 inhibitor is not known in the art. The present disclosure is based, at least in part, on the discovery of new resveratrol derivatives that can act as potent And-1 inhibitors and their use as anti-cancer agents.
In some embodiments, compounds of the present disclosure can be, resveratrol derivatives, And-1 inhibitors, or both. In some embodiments, compounds of the present disclosure can be represented by formula I or a pharmaceutically acceptable salt thereof.
wherein, R1 is independently mono- or poly-substituted with hydrogen, trifluoromethyl, trifluoromethoxy, nitro, cyano, halogen, C1-C10 alkyl, or —OR2, wherein R2 is independently selected from hydrogen or C1-C10 alkyl.
As used herein, an alkyl is an aliphatic alkyl, which may be a linear or branched alkyl, and includes but is not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. The expressions of the form “C1-C10” are intended to include those corresponding groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, for example, “C1-C10 alkyl” refers to an alkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
As used herein, a halogen refers to fluorine, chlorine, bromine or iodine. As used herein, “cyano” refers to a cyano functional group (e.g., —C≡N). As used herein, “nitro” refers to a nitro functional group (e.g., —NO2).
Unless otherwise specified, the structure formula described herein are intended to include all tautomeric, optical, and stereoisomeric forms (e.g., enantiomers, diastereomers, geometric isomers, or conformers). For example, R and S configurations containing asymmetric centers, (Z), (E) isomers of double bonds and conformational isomers of (Z), (E). The individual stereochemical isomers, tautomers or enantiomers, diastereomers or geometric isomers or tautomers, or a mixture of conformers of the compounds herein belong to the scope of the present disclosure.
The term “tautomers” as used herein means that structural isomers with different energies can exceed low energy barriers and thus be converted into each other. For example, proton tautomers (i.e., proton shifts) include interconversions via proton migration, such as 1H-indazole and 2H-indazole, 1H-benzo[d]imidazole and 3H-benzo[d]imidazole. The valence tautomers can include interconversions by reorganization of some bonding electrons.
The pharmaceutically acceptable salts for use herein are not particularly limited, and preferably include: inorganic acid salts, organic acid salts, alkyl sulfonates, and aryl sulfonates; the inorganic acid salts include hydrochloride, hydrobromide, nitrate, sulfate, phosphate, etc.; the organic acid salts include formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, etc.; the alkyl sulfonates include methyl sulfonate, ethyl sulfonate, etc.; and the aryl sulfonates include benzenesulfonate, p-toluenesulfonate, and the like.
In some embodiments, compounds of the present disclosure can be represented by formulas II-XVI or a pharmaceutically acceptable salt thereof, wherein formulas II-XVI include:
or any combination thereof.
The present disclosure also provides embodiments for methods of making any one of the compounds or pharmaceutically acceptable salts thereof as disclosed herein. Methods of making compounds of the present disclosure are further described below with reference to specific examples (e.g. Examples 1-5 herein). It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present disclosure.
In certain embodiments, methods of making any one of the compounds or pharmaceutically acceptable salts thereof as disclosed herein may have one or more of the following steps: (i) reacting a compound represented by Compound A with concentrated hydrochloric acid and paraformaldehyde to obtain a compound represented by Compound B; (ii) reacting a compound represented by Compound B with triphenylphosphonium in an aprotic solvent to obtain a compound represented by Compound C; (iii) producing a compound represented by Compound E from compounds represented by Compound C and Compound D under strong base conditions; (iv) reacting a compound represented by Compound E with trifluoromethanesulfonic anhydride in dichloromethane to obtain a compound represented by Compound F; (v) reacting a compound represented by Compound F with pinacol diboronic acid or diboronic acid under palladium salt catalyst to obtain a compound represented by Compound G; and (vi) reacting a compound represented by Compound G with a reducing agent such and adding acid to obtain a compound represented by Compound H, wherein the reducing agent comprises sodium borohydride, tetrahydroaluminum lithium, or any combination thereof; wherein:
wherein: R1 is independently mono- or poly-substituted with hydrogen, trifluoromethyl, trifluoromethoxy, nitro, cyano, halogen, C1-C10 alkyl, or —OR2, wherein R2 is independently selected from hydrogen or C1-C10 alkyl.
In some embodiments, methods of preparing at least one compound disclosed herein can have one or more steps that require a strong base condition. A strong base condition as used herein can require a step of the method herein be performed in the presence of one or more strong bases. Non-limiting examples of strong bases for use herein can be sodium methoxide, sodium ethoxide, sodium tert-butoxide, sodium isopropoxide, butyl lithium, sodium hydroxide, lithium hydroxide, potassium hydroxide, or any combination thereof. In some examples, methods of preparing at least one compound disclosed herein can include performing step (iii) as disclosed herein in a strong base condition.
In some embodiments, a strong base condition can be a strong alkaline condition. Non-limiting examples of bases for use in a strong alkaline condition can include sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, sodium fluoride, potassium fluoride, or any combination thereof. In some examples, methods of preparing at least one compound disclosed herein may comprise step (v) herein performed under an alkaline condition.
In some embodiments, methods of preparing at least one compound disclosed herein can have one or more steps that require a temperature ranging from about −20° C. to about 100° C., about −10° C. to about 90° C., or about 0° C. to about 80° C. In some embodiments, methods of preparing at least one compound disclosed herein can have one or more steps that require a temperature of about −20° C., about −10° C., about 0° C., about 10° C., about 20° C., about 30° C., about 40° C., about 50° C., about 60° C., about 80° C., about 90° C., or about 100° C. In some examples, methods of preparing at least one compound disclosed herein can have one or more steps that require a temperature of about 0° C. In some other examples, methods of preparing at least one compound disclosed herein can have steps (iv) and (vi) herein performed at about 0° C. In some examples, methods of preparing at least one compound disclosed herein can have one or more steps that require a temperature of about 80° C. In some other examples, methods of preparing at least one compound disclosed herein can have steps (iii) and (v) herein performed at about 80° C.
In some embodiments, methods of preparing at least one compound disclosed herein can have one or more steps that utilize at least one palladium salt catalyst. Non-limiting examples of palladium salt catalysts suitable for use herein can include palladium acetate, palladium chloride, tetrakis(triphenylphosphorus) palladium, bis(acetonitrile) palladium dichloride, bis(triphenylphosphorus) palladium chloride, 1,1′-[bis(diphenylphosphorus)ferrocene]palladium dichloride, bis(benzonitrile)palladium dichloride, 1,1′-[bis(di-tert-butylphosphorus)ferrocene] Palladium dichloride, bis(tricyclohexylphosphorus) palladium dichloride, bis(o-toluene) palladium dichloride, or any combination thereof. In some examples, methods of preparing at least one compound disclosed herein can utilize a palladium salt catalyst in at least step (v) as disclosed herein.
In some embodiments, methods of preparing at least one compound disclosed herein can have one or more steps that utilize at least one polar solvent. Non-limiting examples of polar solvents for use herein can include N, N-dimethylformamide, dimethylsulfoxide, acetonitrile, acetone, methyl ethyl ketone, 1,4-dioxane, water, or any combination thereof. In some examples, methods of preparing at least one compound herein can utilize at least one polar solvent in step (v) as disclosed herein.
One of skill in the art would understand that the methods of making compounds disclosed herein can be modified to increase yield, purity, volume, solubility, crystallinity, and the like.
II. Pharmaceutical Compositions
In some embodiments, compounds disclosed herein for use according to the methods herein described may be provided per se or as part of a pharmaceutical composition, where compounds can be mixed with suitable carriers or excipients. In some embodiments, compositions disclosed herein can include one or more compounds disclosed herein. In some other embodiments, compositions disclosed herein can include one or more compounds disclosed herein and/or one or more anticancer drugs (e.g., one or more platinum-based chemotherapeutics). In certain embodiments, these compounds can be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a cancer (e.g., a tumor) in a subject.
In some embodiments, compositions disclosed herein can include at least one compound represented by formula III or a salt thereof as disclosed herein. In some embodiments, compositions disclosed herein can include at least one compound represented by formula III or a salt thereof as disclosed herein and at least one anticancer drug. In some embodiments, compositions disclosed herein can include at least one compound represented by formula III or a salt thereof as disclosed herein and at least one platinum-based chemotherapeutic. In some embodiments, compositions disclosed herein can include at least one compound represented by formula III or a salt thereof as disclosed herein and cisplatin.
In some embodiments, compositions disclosed herein can include at least (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol. In some embodiments, compositions disclosed herein can include at least (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol and at least one anticancer drug. In some embodiments, compositions disclosed herein can include at least (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol and at least one platinum-based chemotherapeutic. In some embodiments, compositions disclosed herein can include at least (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol and cisplatin.
As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein (e.g., And-1 inhibitors) with other chemical components such as physiologically suitable carriers and excipients. A purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term “active ingredient” refers to one or more of the compounds (e.g., And-1 inhibitors) disclosed herein, one or more anticancer drugs (e.g., platinum-based chemotherapeutics), or a combination thereof.
(i) Pharmaceutically Acceptable Carriers and Excipients
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier”, which may be interchangeably used, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
In various embodiments, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer's solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, anti-oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, and combinations thereof. Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in R
In various embodiments, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In other embodiments, any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art.
In various embodiments, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of polymers as suspending agent(s) by total weight of the composition.
In various embodiments, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of the composition may be increased by the addition of one or more gelling or thickening agents. In other aspects, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In still other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of gelling or thickening agent(s) by total weight of the composition. In yet other aspects, suitable thickening agents can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or combinations thereof. In some embodiments, suitable thickening agent may be carboxymethylcellulose.
In various embodiments, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more agents by total weight of the composition. In other aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelf-life and/or other property of the muscarinic antagonist composition of the present disclosure. In s aspects, additives will be biocompatible, and will not be harsh, abrasive, or allergenic.
In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more acidifying agents by total weight of the composition.
In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more alkalizing agents by total weight of the composition.
In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more antioxidants by total weight of the composition.
In other embodiments, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In another aspect, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In other aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some embodiments, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In other embodiments, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6. In a preferred embodiment, compositions disclosed herein may have a pH greater than about 6.8.
In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition.
In other embodiments, pharmaceutical compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In other aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In still other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition.
In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition.
In other embodiments, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm/L). Osmolarity may be measured using methods commonly known in the art. In preferred embodiments, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 280 mOsm/L to about 370 mOsm/L or about 250 mOsm/L to about 320 mOsm/L. In other aspects, a composition herein may have an osmolality ranging from about 100 mOsm/kg to about 1000 mOsm/kg, from about 200 mOsm/kg to about 800 mOsm/kg, from about 250 mOsm/kg to about 500 mOsm/kg, or from about 250 mOsm/kg to about 320 mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about 280 mOsm/kg to about 320 mOsm/kg. In some embodiments, a pharmaceutical composition described herein has an osmolarity of about 100 mOsm/L to about 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 250 mOsm/L to about 320 mOsm/L, or about 280 mOsm/L to about 320 mOsm/L. In still other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition.
(ii) Dosage Formulations
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as, intravenous, intraperitoneal, intranasal injections.
One may administer the pharmaceutical compositions herein in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some embodiments, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, a pharmaceutical composition disclosed herein can administered to the human patient via at least two administration routes. In some examples, the combination of administration routes by be intramuscular injection and intravenous injection; subcutaneous injection and intravenous injection; intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and intra-parenchymal injection and intravenous injection. In some embodiments, a pharmaceutical composition disclosed herein can administered to a cancer cell, a tumor, or both. In some embodiments, a pharmaceutical composition disclosed herein can administered to a cancer cell, a tumor, or both by introducing the cancer cell, the tumor, or both to a pharmaceutical composition disclosed herein.
Pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present disclosure may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients (e.g., And-1 inhibitors and/or anticancer drugs) into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, tone or more active ingredients (e.g., And-1 inhibitors and/or anticancer drugs) may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
Pharmaceutical compositions suitable for use in context of the present disclosure can include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients (e.g., And-1 inhibitors and compounds disclosed herein) effective to prevent, slow, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in T
Dosage amount and interval may be adjusted individually to brain or blood levels of the active ingredients and/or one of its metabolites are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Effective doses may be extrapolated from dose-responsive curves derived from in vitro or in vivo test systems.
III. Methods of Use
Embodiments of the instant disclosure relate to novel methods and compositions for treating cancers, tumors, or any combination thereof resistant to or suspected of becoming resistant to one or more anticancer drugs/therapies. As used herein an “anticancer drug” refers to any drug with an intended use for the treatment of malignant, or cancerous disease. Anticancer therapy refers to a treatment regimen for the treatment of malignant, or cancerous disease. Non-limiting examples of anticancer therapies can include administration of an anticancer drug, radiation, surgical methods, and the like. Anticancer drugs can be classified into three groups: cytotoxic drugs, hormones, and signal transduction inhibitors. Cytotoxic anticancer drugs suitable for use herein can include, but are not limited to: alkylating agents (e.g., nitrogen mustards and nitrosoureas); antimetabolites (e.g., folate antagonists, purine and pyrimidine analogues); antibiotics and other natural products (e.g., anthracyclines and vinca alkaloids); antibodies that improve drug specificity, and other generally cytotoxic drugs. In some embodiments, anticancer drugs herein can refer to platinum-based chemotherapeutics.
In some embodiments, a tumor subjected to the methods and compositions disclosed herein can be a solid tumor. In some embodiments, compositions and methods disclosed herein can re-sensitize or sensitize a tumor in a subject to one or more anticancer drugs (e.g., platinum-based chemotherapies). In some embodiments, compositions and methods disclosed herein can re-sensitize or sensitize a tumor in a subject to one or more anticancer drugs to reduce costs, improve outcome and reduce or eliminate patient exposure to an anticancer therapy without significant effect. In some embodiments, a subject can have an anticancer drug resistant cancer or be suspected of developing such a cancer where additional agents can be administered to re-sensitize or sensitize the cancer in a subject.
In some embodiments, a subject can have an anticancer drug resistant tumor or be suspected of developing such a tumor where additional agents can be administered to re-sensitize or sensitize a tumor in a subject wherein the tumor can include a solid tumor. In some embodiments, a solid tumor can be an abnormal mass of tissue that is devoid of cysts or liquid regions within the tumor. In some embodiments, solid tumors can be benign (not progressed to a cancer), a malignant or metastatic tumor. In some embodiments, a solid tumor herein can be a malignant cancer that has metastasized. In other embodiments, solid tumors contemplated herein can include, but are not limited to, sarcomas, carcinomas, lymphomas, gliomas or a combination thereof. In accordance with some embodiments herein, tumors resistant to anticancer drugs (e.g., platinum-based chemotherapies) can include, but are not limited to, a testicular tumor, ovarian tumor, cervical tumor, a kidney tumor, bladder tumor, head-and-neck tumor, liver tumor, stomach tumor, lung tumor, endometrial tumor, esophageal tumor, breast tumor, cervical tumor, central nervous system tumor, germ cell tumor, prostate tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, neuroblastoma, sarcoma, multiple myeloma, melanoma, mesothelioma, osteogenic sarcoma or a combination thereof. In some embodiments, a targeted tumor contemplated herein can include a solid tumor such as an ovarian tumor, a breast tumor, or any combination thereof.
Some standards of care in the art for solid tumors include combination therapies but there has been limited success with combination therapies. Such combinations can lead to toxicity of the drugs, significant number of patients that are ineligible for cisplatin-based therapies, and a relatively small therapeutic survival benefit of about 5-15% when these combinations are used. Embodiments disclosed herein are designed to avoid such outcomes as there is a need in the art for new combination therapies that are equally or more effective than the current standard of care, and that offer patients a much more tolerable chemotherapeutic regimen. In some embodiments, methods of administering a combination therapy herein may include administration of any one of the compounds disclosed herein and at least one anticancer drug. In some embodiments, methods of administering a combination therapy herein may include administration of any one of the compounds disclosed herein and at least one anticancer drug wherein the compound and the anticancer drug are formulated into a single dosage form. In some embodiments, methods of administering a combination therapy herein may include administration of any one of the compounds disclosed herein and at least one anticancer drug simultaneously, subsequently, or at dosage intervals determined by one of skill in the art wherein the compound and the anticancer drug are each formulated into individual dosage forms.
In some embodiments, methods of administering any one of the compounds disclosed herein and at least one anticancer drug can have an additive effect on the efficacy of the compound (e.g., And-1 inhibitor), on the efficacy the anticancer drug, or on the efficacy both. In some embodiments, methods of administering any one of the compounds disclosed herein and at least one anticancer drug can have a synergistic effect on the efficacy of the compound (e.g., And-1 inhibitor), on the efficacy the anticancer drug, or on the efficacy both.
Studies have been performed and are currently continuing to identify biomarkers of anticancer drug-resistant cancers such as platinum-resistant cancers. Therapeutically actionable targets in treatment-resistant tumors have been hampered for example, by reliance on using simplistic techniques with tumor-derived cell line models. Embodiments of the present disclosure provides methods and compositions for treating cancers and tumors resistant to anticancer drugs (e.g., platinum-based chemotherapy) by, for example, identifying a subject having tumors with increased expression of And-1 and/or increased phosphorylation of And-1, and/or administering an effective amount of at least one compound (e.g., And-1 inhibitor) disclosed herein in combination with at least one anticancer drug (e.g., platinum-based chemotherapeutic) to the subject for treating a solid tumor depending on whether or not the subject was identified as an And-1 overexpresser, a phosphorylated And-1 protein overexpresser, or a combination thereof.
In some embodiments, compositions of use herein can include one or more compounds (e.g., And-1 inhibitors) or salts thereof. In some embodiments, concentrations of compounds herein or salts thereof for use in therapies disclosed herein can include about 1 mg to about 200 mg, about 5 mg to about 150 mg, about 10 mg to about 120 mg, about 20 mg about 30 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg.
In some embodiments, compositions of use herein can include (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol In some embodiments, concentrations of (E)-5-(3,4-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol for use in therapies disclosed herein can include about 1 mg to about 200 mg, about 5 mg to about 150 mg, about 10 mg to about 120 mg, about 20 mg about 30 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg.
In certain embodiments, compounds herein or salts thereof can be administered to a subject alone or in combination with an anticancer drug, daily, every other day, twice weekly, every other day, every other week, weekly or monthly or other suitable dosing regimen.
In some embodiments, other compositions which can be administered alone or in combination with an And-1 inhibitor-containing composition can include at least one anticancer drug, e.g., a platinum-based chemotherapeutic. As used herein, a “platinum-based chemotherapeutic” is a chemotherapeutic that is an organic compound which contains platinum as an integral part of the molecule. In some embodiments, compositions of use herein can contain one or more platinum-based chemotherapeutics including, but not limited to, cisplatin, carboplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin or a combination thereof. In some embodiments, a platinum-based chemotherapeutic can be administered separately from the compounds disclosed herein. In some embodiments, compositions containing a platinum-based chemotherapeutic of use herein can contain a concentration of the platinum-based chemotherapeutic at about 1 mg/ml to about 100 mg/ml (e.g., about 1 mg/ml, about 5 mg/ml, about 10 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 80 mg/ml, about 100 mg/ml). In some embodiments, the platinum-based chemotherapeutic or salt thereof or derivative thereof includes cisplatin. In certain embodiments, platinum-based chemotherapeutic agents can be administered to a subject alone or in combination with at least one of the compounds disclosed herein (e.g., And-1 inhibitors), daily, every other day, twice weekly, every other day, every other week, weekly or monthly or other suitable dosing regimen.
In certain embodiments, compositions disclosed herein can treat and/or prevent cancer in a subject in need. In some embodiments, compositions disclosed herein can impair tumor growth compared to tumor growth in an untreated subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be stopped following treatment with compositions disclosed herein. In other embodiments, tumor growth can be impaired at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted outcome. In other words, tumors in subject treated using a composition of the disclosure have tumors that grow at least 5% less (or more as described above) when compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be impaired at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be impaired at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated subject with identical disease condition and predicted outcome.
In some embodiments, treatment of tumors with compositions disclosed herein can result in a shrinking of a tumor in comparison to the starting size of the tumor. In some embodiments, tumor shrinking is at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% (meaning that the tumor is completely gone after treatment) compared to the starting size of the tumor.
In various embodiments, compositions disclosed herein can improve patient life expectancy compared to the cancer life expectancy of an untreated subject with identical disease condition and predicted outcome. As used herein, “patient life expectancy” is defined as the time at which 50 percent of subjects are alive and 50 percent have passed away. In some embodiments, patient life expectancy can be indefinite following treatment with a composition disclosed herein. In other aspects, patient life expectancy can be increased at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, patient life expectancy can be increased at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, patient life expectancy can be increased at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated patient with identical disease condition and predicted outcome.
In some embodiments, the methods of the present disclosure increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, and/or tumor burden or load or reduce the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels prior to treatment or in a control subject. In some embodiments, reduction is measured by comparing cell proliferation, tumor growth, and/or tumor volume in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating a cancer in a subject allows one or more symptoms of the cancer to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, before, during, and after the administration of the pharmaceutical composition, cancerous cells and/or biomarkers in a subject are measured in a biological sample, such as blood, serum, plasma, urine, peritoneal fluid, and/or a biopsy from a tissue or organ. In some embodiments, the methods include administration of the compositions of the invention to reduce tumor volume, size, load or burden in a subject to an undetectable size, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the subject's tumor volume, size, load or burden prior to treatment. In other embodiments, the methods include administration of the compositions of the invention to reduce the cell proliferation rate or tumor growth rate in a subject to an undetectable rate, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate prior to treatment.
In some embodiments, a subject to be treated by any of the methods and/or compositions herein can present with one or more cancerous solid tumors, metastatic nodes, of a combination thereof. In some embodiments, a subject herein can have a cancerous tumor cell source that can be less than about 0.2 cm3 to at least about 20 cm3 or greater, at least about 2 cm3 to at least about 18 cm3 or greater, at least about 3 cm3 to at least about 15 cm3 or greater, at least about 4 cm3 to at least about 12 cm3 or greater, at least about 5 cm3 to at least about 10 cm3 or greater, or at least about 6 cm3 to at least about 8 cm3 or greater.
In various embodiments, the compositions disclosed herein can be effective for treating at least one tumor cell in a solid tumor from a subject in need. In some embodiments, the amount of viable tumor cells is reduced by at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome.
In various embodiments, the compositions disclosed herein can reduce the amount of viable tumor cells by at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome.
Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the compositions disclosed herein to a subject, depending upon the type of disease to be treated or the site of the disease. In some embodiments, compositions herein can be administered to a subject by intravenous infusion by subcutaneous administration, by inhalation, by intranasal administration or other mode of administration. In some embodiments, compositions herein can be administered to a subject orally.
In some embodiments, any of the methods disclosed herein can further include monitoring occurrence of one or more adverse effects in the subject. Exemplary adverse effects include, but are not limited to, hepatic impairment, hematologic toxicity, neurologic toxicity, cutaneous toxicity, gastrointestinal toxicity, or a combination thereof. When one or more adverse effects are observed, the method disclosed herein can further include reducing or increasing the dose of one or more of the disclosed compounds (e.g., And-1 inhibitors), the dose of one or more anticancer drugs (e.g., platinum-based chemotherapeutics) or both depending on the adverse effect or effects in the subject. For example, when a moderate to severe hepatic impairment is observed in a subject after treatment, compositions of use to treat the subject can be reduced in concentration or frequency of dosing with one or more disclosed compounds the dose or frequency of the platinum-based chemotherapeutic can be adjusted (e.g., cisplatin) or a combination thereof.
In some embodiments, one or more disclosed compounds (e.g., And-1 inhibitors) herein can be administered concurrently with the one or more anticancer drugs (e.g., platinum-based chemotherapeutics) by the same or different modes of administration. In some embodiments, one or more disclosed compounds (e.g., And-1 inhibitors) herein can be administered before, during or after the one or more anticancer drugs. In other embodiments, the one or more anticancer drugs can be administered systemically. In certain embodiments, the one or more anticancer drugs (e.g., platinum-based chemotherapeutics) can be administered locally directly to one or more tumors in the subject. In some embodiments, the one or more anticancer drugs can be administered by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intrasynovial, intrathecal, intratumoral, oral, inhalation or topical routes. In other embodiments, the one or more anticancer drugs can be administered to the subject by intravenous infusion.
An effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, systemically or locally. In some embodiments, one or more disclosed compounds (e.g., And-1 inhibitors) can be administered by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intrasynovial, intrathecal, intratumoral, oral, inhalation or topical routes. In some embodiments, one or more disclosed compounds (e.g., And-1 inhibitors) can be administered orally.
In some embodiments, dosages for one or more disclosed compounds (e.g., And-1 inhibitors) and/or one or more anticancer drugs (e.g., platinum-based chemotherapeutics) as described herein are determined empirically in individuals who have been given one or more administration(s) of the one or more compounds herein and/or one or more platinum-based chemotherapeutics. Individuals are given incremental dosages of the one or more one or more disclosed compounds (e.g., And-1 inhibitors) and/or one or more anticancer drugs. To assess efficacy of the one or more disclosed compounds (e.g., And-1 inhibitors) and/or one or more anticancer drugs (e.g., platinum-based chemotherapeutics), an indicator of the disease/disorder can be followed.
In some embodiments, methods herein of treating a cancer with one or more disclosed compounds (e.g., And-1 inhibitors) and/or one or more platinum-based chemotherapeutics can further include treating a subject with at least one additional anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery. In some embodiments, an additional anticancer drug can be administered to the subject including, but not limited to, one or more of gemcitabine, methotrexate, vinblastine, and/or adriamycin. In some embodiments, methods herein can be administered to a subject who has completed at least one anticancer therapy or who is currently undergoing at least anticancer therapy. In some embodiments, methods herein can be administered to a subject having received and/or currently receiving at least one immune-modulatory agent. Non-limiting examples of such immune-modulatory agents include, but are not limited to, anti-PD1, anti-PD-L1, anti-CTLA-4, anti-OX40, anti-CD137, etc. Non-limiting examples of PD-1 inhibitors include, but are not limited to, anti-PD-1 antibodies, such as pembrolizumab, nivolumab, and cemiplimab. Non-limiting examples of PD-L1 inhibitors can include atezolizumab, durvalumab, and avelumab. A non-limiting example of a CTLA-4 inhibitor is the anti-CTLA-4 antibody ipilimumab. In some embodiments, an immunomodulatory agent can be one or more inhibitors that target a checkpoint molecule selected from CD40, GITR, LAG-3, OX40, TIGIT and TIM-3. In some embodiments, the additional one or more anticancer drugs can be one or more chemotherapeutics. In some examples, chemotherapeutics for use herein can include an antimetabolite, a microtubule inhibitor, or a combination thereof. Antimetabolites can include, for example, folic acid antagonist (e.g., methotrexate) and nucleotide analogs such as pyrimidine antagonist (e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine), purine antagonist (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitor (e.g., cladribine, fludarabine and pentostatin).
In some embodiments, methods of treatment with at least one or more disclosed compounds (e.g., And-1 inhibitors) and/or one or more anticancer drugs (e.g., platinum-based chemotherapeutics) can depend on the cancer type, grade of cancer, stage or cancer or a combination thereof. In some embodiments, methods of treatment with at least one or more disclosed compounds (e.g., And-1 inhibitors) and/or one or more anticancer drugs (e.g., platinum-based chemotherapeutics) can depend on the stage of cancer as determined by the TNM system wherein “T” stands for tumor, “N” stands for node, and “M” stands for metastasis. When applying the TNM system, the following are considered: Tumor (T)—How large is the primary tumor? Where is it located?; Node (N)—Has the tumor spread to the lymph nodes? If so, where and how many?; and Metastasis (M)—Has the cancer spread to other parts of the body? If so, where and how much? In some embodiments, methods of treatment with at least one or more disclosed compounds (e.g., And-1 inhibitors) and/or one or more anticancer drugs (e.g., platinum-based chemotherapeutics) can depend on the stage of bladder cancer. One of skill in the art (i.e., a physician) can assign the stage of the cancer in a subject by combining the T, N, and M classifications.
In some embodiments, methods for measuring And-1 levels in a targeted tumor are disclosed where And-1 can be used as at least one biomarker, for example, to select or identify patients or subjects in need of aminopeptidase adjustment. In certain embodiments, And-1 levels can determine tumor burden, disease progression or projected response to treatment.
As used herein, “biomarker” can mean a distinctive biological or biologically derived indicator of a process, event or conditions. In some embodiments, the biomarker is a gene or gene product (e.g., a polypeptide). In some embodiments, a combination of biomarkers can be used as an indicator of a process, event or condition. In some embodiments, a combination of biomarkers used as an indicator of a process, event or condition can encompass at least two biomarkers. In some embodiments, a combination of biomarkers used as an indicator of a process, event or condition can encompass at least two biomarkers wherein one of the at least two biomarkers can be And-1.
As used herein, “predictive biomarker” can refer to a biomarker that can be used in advance of therapy to estimate the likelihood or predictability of response to a given therapeutic agent (e.g., anticancer drug) or class of therapeutic agents. In some embodiments, a combination of predictive biomarkers can be used in advance of therapy to estimate the likelihood or predictability of response to a given therapeutic agent or class of therapeutic agents. In some embodiments, a combination of predictive biomarkers used in advance of therapy to estimate the likelihood or predictability of response to a given therapeutic agent or class of therapeutic agents can have at least two predictive biomarkers. In some embodiments, a combination of predictive biomarkers used in advance of therapy to estimate the likelihood or predictability of response to a given therapeutic agent or class of therapeutic agents can have at least one predictive biomarkers wherein one of the predictive biomarkers can be And-1.
As used herein, the term “biological sample” can mean a sample obtained from a subject. A suitable biological sample can be obtained from a subject as described herein via routine practice. Non-limiting examples of biological samples include fluid samples such as blood (e.g., whole blood, plasma, or serum), urine, and saliva, and solid samples such as tissue (e.g., skin, lung, or nasal) and feces. Such samples can be collected using any method known in the art or described herein, e.g., buccal swab, nasal swab, venipuncture, biopsy, urine collection, or stool collection. In some embodiments, the biological sample can be a blood sample. In some other embodiments, the blood sample is a serum sample or a plasma sample. In some embodiments, a biological sample can be derived from a tissue and/or tumor biopsy collected from the subject e.g., patient-derived organotypic tumor spheroids prepared as described herein.
In some embodiments, methods herein of measuring And-1 levels in a biological sample of subject can be measured by routine practice. Methods for detecting and/or assessing an amount of gene expression of And-1 in a biological sample are well known in the art, and all suitable methods for detecting and/or assessing an amount of gene expression levels known to one of skill in the art are contemplated within the scope of the invention. In some embodiments, gene expression of And-1 in a biological sample can be measured by high-density expression array, DNA microarray, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), real-time quantitative reverse transcription PCR (qRT-PCR), digital droplet PCR (ddPCR), serial analysis of gene expression (SAGE), Spotted cDNA arrays, GeneChip, spotted oligo arrays, bead arrays, RNA Seq, tiling array, northern blotting, hybridization microarray, in situ hybridization, or a combination thereof. In some aspects, gene expression of And-1 as disclosed herein can be measured by any known or future method suitable to assess gene expression.
Methods for detecting and/or assessing an amount of protein expression of And-1 and phosphorylated And-1 in a biological sample are well known in the art, and all suitable methods for detecting and/or assessing an amount of protein expression levels known to one of skill in the art are contemplated within the scope of the invention. In some embodiments, protein expression of And-1 in a biological sample can be measured by western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, HPLC, flow cytometry, fluorescence-activated cell sorting (FACS), liquid chromatography-mass spectrometry (LC/MS), immunoelectrophoresis, translation complex profile sequencing (TCP-seq), protein microarray, protein chip, capture arrays, reverse phase protein microarray (RPPA), two-dimensional gel electrophoresis or (2D-PAGE), functional protein microarrays, electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), or a combination thereof. In some aspects, protein expression of And-1 and phosphorylated And-1 as disclosed herein can be measured by any known or future method suitable to assess protein expression.
In some embodiments, a subject can be a human patient having an elevated level of And-1 relative to a control level. In some embodiments, a subject can be a human patient having an elevated level of phosphorylated And-1 protein relative to a control level. A control level can refer to the level of And-1 or the level of phosphorylated And-1 protein in a matched sample of a subject of the same species (e.g., human) who are free of the solid tumor. In some examples, the control level represents the level of And-1 or the level of phosphorylated And-1 protein in healthy subjects.
IV. Kits
In certain embodiments, the present disclosure provides kits for use in treating or alleviating a solid tumor described herein. Such kits can include one or more containers including one or more one or more disclosed compounds (e.g., And-1 inhibitors), e.g., any of those described. In some embodiments, kits can include one or more containers including one or more one or more disclosed compounds (e.g., And-1 inhibitors), e.g., any of those described and one or more platinum-based chemotherapeutic described herein (e.g., a cisplatin). In other embodiments, kits can include a control And-1 sample for assessing level of And-1 in a sample from a subject.
In some embodiments, the kits herein can include instructions for use in accordance with any of the methods described herein. The included instructions can have a description of administration of the one or more disclosed compounds (e.g., And-1 inhibitors), the one or more anticancer drugs (e.g., platinum-based chemotherapeutics), to treat, delay the onset, or alleviate a target disease as those described herein, or a combination thereof. In some embodiments, the kit can further include a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease, e.g., applying the diagnostic method as described herein. In still other embodiments, the instructions can have a description of administering any one of the compositions described herein to an individual at risk of the target disease.
In some embodiments, kit instructions relating to the use of one or more one or more disclosed compounds (e.g., And-1 inhibitors), one or more anticancer drugs (e.g., platinum-based chemotherapeutics) described herein (e.g., a cisplatin), or a combination thereof can generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the solid tumor. In some embodiments, instructions are provided for practicing any of the methods described herein. In some embodiments, instructions are provided for assessing And-1 as a biomarker of disease (e.g., cancer).
The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. In some embodiments, a kit has a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is at least one or more disclosed compounds (e.g., And-1 inhibitors).
In some embodiments, kits herein can optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.
EXAMPLESThe following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
Example 1: Synthesis of 5-(chloromethyl)-2-hydroxybenzaldehyde (B)A solution of paraformaldehyde (24.40 grams (g), 0.20 moles (mol)) in concentrated hydrochloric acid (80 milliliters (mL)) was stirred at room temperature for 10 minutes, and then salicylaldehyde (17.95 g, 0.60 mol) was added dropwise over 30 minutes. The reaction solution was stirred at room temperature for 24 hours to give a white solid. Filtration, washing the filter cake with cold water, and drying to obtain a crude product. Recrystallization from n-hexane (310 mL) gave white crystals (16.80 g, 49.4%). 1H NMR (400 megahertz (MHz), DMSO-d6) δ 10.91 (s, 1H), 10.27 (s, 1H), 7.72 (d, J=2.4 Hz, 1H), 7.59 (dd, J=8.5, 2.4 Hz, 1H), 7.03 (d, J=8.5 Hz, 1H), 4.76 (s, 2H).
Example 2: Synthesis of (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride (C)5-(chloromethyl)-2-hydroxybenzaldehyde (15.50 g, 91.2 mmol) and triphenylphosphine (30.98 g, 118.1 mmol) were dissolved in 220 mL of dry acetonitrile. The reaction solution was stirred at 80° C. for 3 hours under a nitrogen atmosphere. The acetonitrile was evaporated to dryness under reduced pressure, and the residue was washed with petroleum ether (100 mL×3) to afford white powder (38.52 g, 97.8%).
Example 3: Synthesis of (E)-5-(3,5-dichlorostyryl)-2-hydroxybenzaldehyde (E1)Sodium (0.19 g, 8.26 mmol) was dissolved in absolute ethanol (30 ml) under nitrogen. The phosphonium salt (1.20 g, 2.78 mmol) was added to this sodium ethoxide solution until the reaction mixture became dark yellow, and 3,5-dichlorobenzaldehyde (0.57 g, 3.26 mmol) was added. The reaction solution was stirred at 75° C. for 3 hours. The reaction was withdrawn, cooled to room temperature, clarified and then turbid with water, and the pH was adjusted to be acidic with dilute hydrochloric acid to precipitate a yellow solid. Filtration, drying and recrystallization from tetrahydrofuran afforded yellow crystals (0.387 g, 44.4%). 1H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 10.31 (s, 1H), 7.87 (d, J=2.2 Hz, 1H), 7.79 (dd, J=8.7, 2.3 Hz, 1H), 7.67 (d, J=1.8 Hz, 2H), 7.46 (d, J=16.5 Hz, 1H), 7.45 (t, J=1.8 Hz, 1H), 7.13 (d, J=16.5 Hz, 1H), 7.06 (d, J=8.6 Hz, 1H).
By performing the steps described in Example 3, the following intermediates were prepared:
1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 10.31 (s, 1H), 7.87 (d, J=1.9 Hz, 1H), 7.80 (dd, J=8.6, 1.9 Hz, 1H), 7.49-7.36 (m, 3H), 7.32 (d, J=16.4 Hz, 1H), 7.14 (d, J=16.4 Hz, 1H), 7.07 (dd, J=14.4, 6.6 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 191.56, 164.30, 161.89, 161.09, 140.39, 140.31, 134.54, 130.99, 130.91, 129.28, 128.79, 127.59, 126.19, 126.16, 123.15, 123.13, 122.87, 118.29, 114.52, 114.30, 112.88, 112.67;
1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.31 (s, 1H), 7.87 (d, J=2.0 Hz, 1H), 7.81 (dd, J=8.6, 2.1 Hz, 1H), 7.68 (s, 1H), 7.54 (d, J=7.8 Hz, 1H), 7.35 (dt, J=17.0, 7.9 Hz, 3H), 7.14 (d, J=16.5 Hz, 1H), 7.05 (d, J=8.6 Hz, 1H);
1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 10.30 (s, 1H), 7.84 (d, J=2.2 Hz, 1H), 7.80 (dd, J=8.6, 2.3 Hz, 1H), 7.44-7.34 (m, 2H), 7.29-7.17 (m, 2H), 7.14-7.01 (m, 3H), 2.32 (s, 3H);
1H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 10.31 (s, 1H), 7.93-7.75 (m, 2H), 7.39-7.22 (m, 2H), 7.14 (dd, J=16.7, 12.2 Hz, 3H), 7.04 (d, J=8.6 Hz, 1H), 6.88-6.79 (m, 1H), 3.79 (s, 3H);
1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 10.31 (s, 1H), 7.86 (s, 1H), 7.84-7.78 (m, 1H), 7.59 (d, J=7.7 Hz, 2H), 7.37 (t, J=7.6 Hz, 2H), 7.30-7.20 (m, 2H), 7.14 (d, J=16.5 Hz, 1H), 7.04 (d, J=8.6 Hz, 1H);
1H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 10.31 (s, 1H), 7.95 (s, 1H), 7.90 (t, J=4.3 Hz, 2H), 7.83 (dd, J=8.7, 2.3 Hz, 1H), 7.65-7.55 (m, 2H), 7.44 (d, J=16.5 Hz, 1H), 7.26 (d, J=16.5 Hz, 1H), 7.06 (d, J=8.6 Hz, 1H);
1H NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H), 10.31 (s, 1H), 7.87 (d, J=1.9 Hz, 2H), 7.80 (dd, J=8.6, 2.3 Hz, 1H), 7.65-7.52 (m, 2H), 7.37 (d, J=16.5 Hz, 1H), 7.13 (d, J=16.5 Hz, 1H), 7.05 (d, J=8.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 191.43, 161.22, 138.63, 134.57, 131.94, 131.13, 130.08, 129.72, 128.62, 128.24, 127.64, 126.74, 124.79, 122.88, 118.30;
1H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 10.32 (s, 1H), 8.29 (s, 2H), 7.93 (d, J=2.0 Hz, 1H), 7.91 (s, 1H), 7.83 (dd, J=8.6, 2.1 Hz, 1H), 7.67 (d, J=16.6 Hz, 1H), 7.37 (d, J=16.5 Hz, 1H), 7.07 (d, J=8.6 Hz, 1H);
1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.31 (s, 1H), 7.89 (d, J=2.2 Hz, 1H), 7.84-7.74 (m, 3H), 7.59-7.48 (m, 2H), 7.26 (d, J=16.5 Hz, 1H), 7.06 (d, J=8.6 Hz, 1H);
1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 10.31 (s, 1H), 7.98 (d, J=7.9 Hz, 1H), 7.85 (d, J=2.2 Hz, 1H), 7.80 (dd, J=8.6, 2.3 Hz, 1H), 7.74 (d, J=7.8 Hz, 1H), 7.69 (t, J=7.6 Hz, 1H), 7.48 (t, J=7.6 Hz, 1H), 7.35 (d, J=16.1 Hz, 1H), 7.24 (dd, J=16.2, 2.1 Hz, 1H), 7.08 (d, J=8.6 Hz, 1H);
1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 10.31 (s, 1H), 7.90 (d, J=2.2 Hz, 1H), 7.85 (dd, J=8.6, 2.2 Hz, 1H), 7.79 (d, J=8.2 Hz, 2H), 7.71 (d, J=8.3 Hz, 2H), 7.43 (d, J=16.5 Hz, 1H), 7.24 (d, J=16.5 Hz, 1H), 7.05 (d, J=8.6 Hz, 1H); and
1H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 10.31 (s, 1H), 7.89 (d, J=2.2 Hz, 1H), 7.82 (dd, J=8.6, 2.3 Hz, 1H), 7.66-7.56 (m, 2H), 7.49 (t, J=8.0 Hz, 1H), 7.38 (d, J=16.5 Hz, 1H), 7.21 (t, J=12.3 Hz, 2H), 7.05 (d, J=8.6 Hz, 1H).
Example 4: Synthesis of (E)-4-(3,5-dichlorostyryl)-2-formylphenyl trifluoromethanesulfonate (F1)(E)-5-(3,5-dichlorostyryl)-2-hydroxybenzaldehyde (0.557 g, 1.90 mmol) and pyridine (0.750 g, 9.49 mmol) were dissolved in anhydrous dichloromethane (25 mL), and the mixture was stirred at 0° C. A solution of trifluoromethanesulfonic anhydride (0.810 g, 2.87 mmol) in dichloromethane was added dropwise. After the reaction solution was stirred for 40 minutes in an ice bath, it was monitored by Thin layer Chromatography (TLC) to determine when reactants were consumed and a new product was formed. The reaction mixture was poured into ice water, and the mixture was adjusted to pH 2 with dilute aqueous hydrochloric acid, washed with saturated brine, dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by column chromatography (petroleum ether:ethyl acetate 4:1). A white solid (0.775 g, 96.3%) was obtained. 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.28 (s, 1H), 8.03 (d, J=8.6 Hz, 1H), 7.71 (s, 2H), 7.65 (d, J=8.6 Hz, 1H), 7.60 (d, J=16.5 Hz, 1H), 7.50 (s, 1H), 7.41 (d, J=16.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 189.15, 147.29, 140.62, 138.38, 134.99, 134.27, 131.74, 129.48, 129.21, 128.85, 127.78, 125.79, 123.84, 123.43, 120.24, 117.06, 113.87.
By performing the steps described in Example 4, the following intermediates were prepared:
1H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.39 (d, J=2.3 Hz, 1H), 8.11 (dd, J=8.6, 2.3 Hz, 1H), 7.70 (d, J=8.5 Hz, 1H), 7.63-7.46 (m, 5H), 7.26-7.15 (m, 1H);
1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.33 (d, J=2.3 Hz, 1H), 8.06 (dd, J=8.6, 2.3 Hz, 1H), 7.76 (s, 1H), 7.63 (dd, J=18.5, 8.1 Hz, 2H), 7.53 (d, J=16.5 Hz, 1H), 7.50-7.35 (m, 3H);
1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.34 (d, J=2.3 Hz, 1H), 8.06 (dd, J=8.6, 2.3 Hz, 1H), 7.62 (d, J=8.6 Hz, 1H), 7.52-7.39 (m, 4H), 7.31 (t, J=7.6 Hz, 1H), 7.16 (d, J=7.5 Hz, 1H), 2.35 (s, 3H);
1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.34 (d, J=1.9 Hz, 1H), 8.06 (dd, J=8.6, 2.0 Hz, 1H), 7.64 (d, J=8.5 Hz, 1H), 7.45 (s, 2H), 7.34 (t, J=7.9 Hz, 1H), 7.23 (d, J=7.3 Hz, 2H), 6.99-6.86 (m, 1H), 3.82 (s, 3H);
1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.35 (d, J=2.3 Hz, 1H), 8.07 (dd, J=8.6, 2.3 Hz, 1H), 7.65 (dd, J=14.2, 8.0 Hz, 3H), 7.53-7.39 (m, 4H), 7.34 (t, J=7.3 Hz, 1H);
1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.36 (d, J=2.3 Hz, 1H), 8.09 (dd, J=8.6, 2.3 Hz, 1H), 8.03 (s, 1H), 7.96 (d, J=6.7 Hz, 1H), 7.72-7.54 (m, 5H);
1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.28 (d, J=2.1 Hz, 1H), 8.02 (dd, J=8.6, 2.2 Hz, 1H), 7.90 (d, J=1.4 Hz, 1H), 7.68-7.56 (m, 3H), 7.51 (d, J=16.5 Hz, 1H), 7.41 (d, J=16.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 189.20, 147.11, 138.55, 137.70, 134.14, 132.10, 131.71, 131.33, 130.92, 129.79, 128.83, 128.79, 128.23, 127.42, 123.77, 120.24, 117.05;
1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.47-8.28 (m, 3H), 8.09 (dd, J=8.6, 2.1 Hz, 1H), 8.03 (s, 1H), 7.84 (d, J=16.6 Hz, 1H), 7.69 (dd, J=12.3, 10.5 Hz, 2H);
1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.38 (s, 1H), 8.11 (dd, J=8.6, 1.9 Hz, 1H), 7.87 (d, J=8.0 Hz, 2H), 7.78 (d, J=7.5 Hz, 2H), 7.66 (dd, J=8.5, 2.0 Hz, 1H), 7.64-7.52 (m, 2H); and
1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.35 (d, J=2.3 Hz, 1H), 8.08 (dd, J=8.7, 2.4 Hz, 1H), 7.73-7.62 (m, 3H), 7.60-7.48 (m, 3H), 7.35-7.29 (m, 1H).
Example 5: Synthesis of (E)-5-(3,5-dichlorostyryl)benzo[c][1,2]oxaborol-1(3H)-ol (XIV)B2(Pin)2 (0.870 g, 3.43 mmol) and potassium acetate (0.510 g, 5.19 mmol) were added to dry 1,4-dioxane (18 mL) under nitrogen atmosphere. (E)-4-(3,5-dichlorostyryl)-2-formylphenyl trifluoromethanesulfonate (0.725 g, 1.71 mmol) and Pd(dppf)Cl2 (0.140 g, 0.17 mmol) were then added. The mixture was stirred at 80° C. for two hours. After the dioxane was removed under reduced pressure, the residue was purified by flash column chromatography to obtain a crude product, which was directly used in the next reaction. The crude product was dissolved in a mixed solution of 4 mL methanol and 3 mL tetrahydrofuran, and NaBH4 (0.280 g, 7.41 mmol) was added in portions at 0° C. After the mixture was stirred for one hour, part of the solvent was removed under reduced pressure, 15 mL of water was added, and the pH was adjusted to 3 with dilute hydrochloric acid. After extraction with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The residue was first purified by column chromatography and then recrystallized from toluene to obtain 0.250 g of white solid. The two-step yield was 48.2%. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 7.76 (d, J=7.6 Hz, 1H), 7.72 (d, J=1.7 Hz, 2H), 7.65-7.57 (m, 2H), 7.54 (d, J=16.5 Hz, 1H), 7.50 (s, 1H), 7.33 (d, J=16.4 Hz, 1H), 5.04 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 155.02, 141.25, 139.06, 134.92, 132.22, 131.27, 127.20, 127.15, 126.26, 125.47, 119.81, 70.29.
By performing the steps described in Example 5, the following intermediates were prepared:
1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.75 (d, J=7.3 Hz, 1H), 7.66-7.56 (m, 2H), 7.50 (d, J=10.6 Hz, 1H), 7.46-7.32 (m, 4H), 7.12 (s, 1H), 5.03 (s, 2H);
1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 7.75 (d, J=7.6 Hz, 1H), 7.71 (s, 1H), 7.59 (dd, J=13.9, 6.6 Hz, 3H), 7.46-7.37 (m, 2H), 7.37-7.30 (m, 2H), 5.02 (s, 2H) (
1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 7.74 (d, J=7.6 Hz, 1H), 7.64-7.54 (m, 2H), 7.47-7.38 (m, 2H), 7.33-7.22 (m, 3H), 7.10 (d, J=7.5 Hz, 1H), 5.02 (s, 2H), 2.33 (s, 3H) (
1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.74 (d, J=7.6 Hz, 1H), 7.66-7.55 (m, 2H), 7.37-7.26 (m, 3H), 7.20 (d, J=6.3 Hz, 2H), 6.87 (dd, J=8.0, 1.8 Hz, 1H), 5.02 (s, 2H), 3.80 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.08, 155.06, 139.75, 138.80, 131.24, 130.19, 129.97, 129.21, 126.00, 119.67, 119.46, 114.11, 112.19, 70.29, 55.53;
1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.75 (d, J=7.6 Hz, 1H), 7.60 (dd, J=12.1, 7.7 Hz, 4H), 7.38 (t, J=7.6 Hz, 2H), 7.35-7.24 (m, 3H), 5.01 (s, 2H) (
1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 7.94 (dd, J=12.7, 8.8 Hz, 2H), 7.76 (d, J=7.6 Hz, 1H), 7.62 (dd, J=14.5, 8.9 Hz, 4H), 7.51 (d, J=16.5 Hz, 1H), 7.45 (d, J=16.5 Hz, 1H), 5.03 (s, 2H) (
1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 7.89 (s, 1H), 7.74 (d, J=7.6 Hz, 1H), 7.65-7.54 (m, 4H), 7.43 (d, J=16.5 Hz, 1H), 7.32 (d, J=16.5 Hz, 1H), 5.02 (s, 2H) (
1H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.31 (s, 2H), 7.93 (s, 1H), 7.77 (d, J=7.6 Hz, 1H), 7.71 (d, J=16.5 Hz, 1H), 7.67-7.59 (m, 2H), 7.54 (d, J=16.5 Hz, 1H), 5.03 (s, 2H) (
1H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.00 (d, J=7.8 Hz, 1H), 7.74 (dt, J=20.2, 7.7 Hz, 3H), 7.62 (s, 1H), 7.57 (d, J=7.5 Hz, 1H), 7.54-7.47 (m, 1H), 7.41 (t, J=14.1 Hz, 2H), 5.03 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 155.18, 139.00, 135.91, 133.76, 133.25, 131.44, 128.48, 127.86, 126.39 (q, 2JC-F=29.7 Hz), 126.36, 125.40 (q, 1JC-F=182.7 Hz), 119.74, 70.33;
1H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 7.95-7.31 (m, 9H), 5.02 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 155.05, 141.43, 139.18, 131.76, 131.28, 128.42, 128.17 (q, 2JC-F=31.1 Hz), 127.55, 126.28, 126.01, 119.83, 70.28; and
1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 7.75 (d, J=7.6 Hz, 1H), 7.71-7.58 (m, 4H), 7.53 (t, J=8.0 Hz, 1H), 7.50-7.36 (m, 2H), 7.31-7.24 (m, 1H), 5.03 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 155.06, 149.41, 140.01, 139.35, 131.25, 131.05, 130.96, 128.45, 126.19, 126.08, 120.61 (q, 1JC-F=257.3 Hz), 120.36, 119.69, 119.21, 70.28.
Example 6: Identification of And-1 Inhibitors by High Throughput Screen AssayTo identify And-1 specific pharmacologic inhibitors, a high-throughput screening (HTS) assay was established to identify small molecules that can inhibit And-1 by inducing its degradation. For this assay, two stable cell lines, a HEK293T-And-1-Luc cell expressing And-1 protein fused to a luciferase reporter gene, and a HEK293T-Luc cell expressing luciferase only were generated (
Using a human ovarian cancer cell line (IGROV1 cells), it was confirmed that resveratrol could inhibit And-1 expression with relative high IC50 value (
Next, flow cytometry analysis (specifically, fluorescence-activated cell sorting (FACS)) was performed on propidium-iodide stained IGROV1 cells treated with either DMSO, 1 μM of CH3, 1 μM of BZA, and siAnd-1. FACS analyses indicated that both CH3 and BZA reduced the percentage of cells in the S-phase in a manner similar to those in And-1 depleted cells (
H2AX (H2A histone family member X) becomes phosphorylated on serine 139 (wherein the phosphorylated form is then called 7H2AX) as a reaction on DNA double-strand breaks (DSB). And-1 regulates DSB end resection by establishing a condition or acting as a platform for recruitment of the tumor suppressor protein CtIP—a CtBP (carboxy-terminal binding protein)-interacting protein, to the DSB sites. To determine if the 12 analogs of resveratrol disrupted this complex, an immunofluorescence assay of CtIP co-localization with γH2AX after CH3 or BZA treatment was performed. In brief, IGROV1 cells were cultured on coverslips coated with 0.01% of poly-L-lysine. After treatment, the cells were then washed once with PBS, then pre-extracted with 0.5% Triton X-100 extraction buffer (10 mM PIPES pH 7.0, 100 mM NaCl, 3 mM MgCl2 and 300 mM sucrose) on ice for 5 minutes. After washing with PBS, cells were fixed with 4% paraformaldehyde in PBS for 15 minutes. Cells were then washed and incubated for 10 minutes in blocking buffer (PBS containing 3% BSA and 0.02% Tween-20) and subsequently incubated for 1 hour with a primary antibody at room temperature. Cells were washed three times with PBS-T (PBS containing 0.02% Tween-20) and then incubated with a secondary antibody (e.g., rabbit Alexa Fluor-594 and mouse Alexa Fluor-488). After washing with PBS-T, cells were mounted with Fluoromount G containing DAPI (4′,6-diamidino-2-phenylindole—a fluorescent stain that binds strongly to adenine-thymine-rich regions in DNA). Slides were then imaged using a Nikon Eclipse 80i microscope.
IGROV1 cells treated with DMSO, 1 μM of CH3 or 1 μM of BZA were micro-irradiated and co-immunostained for 7H2AX and CtIP 30 minutes post irradiation. Briefly, cells were labeled with 10 μM BrdU for 48-72 h. Cells were then washed with PBS and covered by a micropore membrane (Isopore 5 μM, Millipore), followed by exposure to UVC lamp at a dose of 30 J/m2 (Hitachi, Germicidal lamp, GL-15). After the exposure, cells were incubated in culture medium for 30 minutes and then subjected to immunofluorescence. For each experiment, 50 cells were counted and the percentage of γH2AX cells exhibiting CtIP foci was determined. Consistently, CH3 and BZA treatment significantly decreased the recruitment of CtIP to DSB sites induced by micro-irradiation (
Homologous recombination (HR) reporter assays were performed to determine HR repair efficiency in cells treated with CH3. HR reporter assays were performed using methods similar to those described in Nakanishi et al., Methods Mol Biol. 2011; 745:283-291, the disclosure of which is incorporated herein in its entirety. Briefly, IGROV1 cells treated with DMSO, 2 μM or 4 μM of CH3, or 2 μM or 4 μM of BZA were subjected to HR reporter assays and the percentage of GFP-positive cells was determined by flow cytometry 48 hours after I-SceI plasmid transfection. HR efficiency was normalized to that obtained from cells treated with DMSO. HR reporter assays showed that inhibition of And-1 by BZA profoundly reduced HR efficiency and delayed DSB repair (
To explore whether And-1 inhibitors directly bind to And-1, cellular thermal shift assays were performed in which a small molecule binds to a protein resulting in thermal stabilization of the protein. Briefly, cells were incubated with MG132 (10 μM), DHS, CH3, CH10 or DMSO for 8 hours. After washing with ice-cold PBS (suppled with Protease Inhibitor Cocktail), cells were aliquoted into PCR tubes (100 μL each) and then incubated at different temperatures (from 25° C. to 71° C.) for 4 minutes. After the cells were frozen and thawed twice using liquid nitrogen, proteins were isolated from the cells after centrifugation and incubated at 70° C. for 10 minutes for analysis by western blotting. From this analysis, it was found that at higher temperatures, And-1 protein was stabilized upon treatment with DHS, CH3 or BZA (
Boron-containing compounds can be visualized using a fluorescent boron sensor DAHMI. To determine the binding specificity of CH3 to And-1, the distribution of CH3 in live cells was examined using DAHMI. As shown in
To identify the CH3-binding region on And-1, the DAHMI system was used in And-1 knockout U20S cells to analyze the binding affinity of CH3 with ectopically expressed And-1 and its truncation mutants, including And-1(1-330) containing the WD40 domain, And-1 (330-1129) containing SepB and HMG domain, And-1 (330-984) containing Sep domain and And-1 (984-1129) containing HMG domain (
Indeed, the thermoshift assay indicated that either the R191A or E18A mutation slightly reduced And-1 thermostability, but mutation of both R191A and E18A significantly impaired the thermostability of And-1 in response to CH3 treatment (
To investigate the molecular mechanism by which And-1 inhibitors induced And-1 degradation, it was first determined whether the ubiquitin-mediated proteasome pathway was involved in CH3-induced And-1 degradation. IGROV1 cells were treated with CH3 in the absence and presence of the 26S proteasome inhibitor MG132 (Benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate). As shown in
Previous studies in yeast cells indicated that the E3 ligase Rtt101 interacts with Ctf4. Since the human analogue of Rtt101 is Cul4, the Cul4 expression in IGROV1 cells was knockeddown by siRNA to determine of Cul4 was the E3 ligase responsible for And-1 degradation. Indeed, knockdown of CUL4B by siRNA dramatically restored And-1 protein levels in cells treated with CH3, indicating that And-1 was a substrate of CUL4B for ubiquitination by CH3 (
Given that the WD40 domain of And-1 mediated the interaction of And-1 with CH3 as shown in the Examples above, it was assumed that And-1 mutants that lack a WD40 domain should not be degraded by CH3. Indeed, the And-1-330-1129 mutant was not degraded in response to CH3 (
Human And-1 exists as a homotrimer mediated by the SepB domain. To determine whether the interaction of CH3 with the WD40 domain can disrupt And-1 polymerization, resulting in exposure of CUL4B binding site on And-1 to CUL4B, both FLAG-And-1 and HA-And-1 were transfected into U2OS cells and the protein-protein interaction was examined in the presence of CH3. As shown in
Cisplatin causes cell death by introducing DNA crosslinks that inhibits DNA replication and transcription. ATR (ataxia telangiectasia and Rad3-related) is an essential DNA damage response (DDR) kinase that phosphorylates And-1 at amino acid residue T826 under replication stress. To explore the physiological significance of And-1 phosphorylation at T826, an antibody that specifically detects the And-1 phosphorylated protein phosphorylated at T826 (referred to herein interchangeably as “phospho-And-1 antibody” or “p-And-1”) was developed. This antibody specifically recognized phosphorylation of And-1 at T826 in cells treated with cisplatin in a time dependent manner (
The expression level of p-And-1 protein was examined the in five paired human ovarian cancer (OC) cell lines, including IGROV1, cisplatin resistant IGROV1 (IGROV1 CR), PEO1, PEO4, OV90, OV90 CR, PEO14, PEO23, OV433 and OV433 CR. Briefly, the human OC PEO1 and PEO4 cells were cultured in RPMI-1640 with 10% Fetal Bovine Serum (FBS). IGROV1 cells were cultured in DMEM with 10% FBS. OV433 cells were cultured in DMEM with 10% FBS. OV90 cells were cultured in the growth medium containing 1:1 MCDB 105 and M199 supplemented with 10% FBS. All the cells were cultured at 37° C. in a humidified incubator containing 5% CO2. Cisplatin resistant (“CR”) cells were generated by first treating wild-type with cisplatin for six cycles (4 hours of cisplatin treatment, followed by release to cisplatin free medium for three weeks). In the next cycle, cisplatin treatment was repeated with an increased concentration of cisplatin. After five months of treatment (6 cycles), cisplatin resistant cell lines IGROV1 CR, OV90 CR and OV433 CR were obtained. Only early-passage (<10 passages) resistant cell lines were used for the Examples herein.
Significantly, p-And-1 levels were increased in all cisplatin resistant cells compared to their sensitive counterparts (
To directly evaluate p-And-1 expression in response to chemotherapy, phosphorylated And-1 protein expression levels in the samples from the same patient before platinum treatment and after acquired drug resistance were examined. The specimens of chemosensitive and matched recurrent or chemoresistant tumor tissues from OC patients were kept as formalin-fixed paraffin-embedded (FFPE) samples. The histological types, disease stages, and cancer cell contents in each FFPE sections were examined by experienced pathologists. Platinum-sensitive was defined as patients who have a total response to platinum-based therapy and no recurrence within 6 months. Platinum resistance means patients who had the recurrence occur within 6 months following the completion of platinum-based therapy.
Strikingly, 7 out of 11 patients showed upregulated p-And-1 protein level after developing platinum drug resistance (
Given that And-1 phosphorylation was positively correlated with cisplatin resistance in OC according the Examples above, it was next determined whether And-1 phosphorylation contributes to cell survival in response to cisplatin. The cell viability to cisplatin in IGROV1, OV90 and mice ID80 cells overexpressed with FLAG-And-1-WT, FLAG-And-1-T826A and phosphomimetic FLAG-And-1-T826E was examined. In brief, cells were seeded in 96-well plate at a density of 3,000-5,000 cells per well and treated with indicated reagents for different concentration. Three days after treatment, the cell viability was measured by Sulforhodamine B (SRB) assay. The IC50 values and CI index were further determined by compusyn software. Every experiment was done in triplicate and repeated three times. Compared to cells transfected with FLAG-And-1-WT, overexpression of FLAG-And-1-T826E significantly increased cell resistance to cisplatin, while overexpression of non-phosphorylatable FLAG-And-1-T826A failed to restore resistance (
To determine if increased And-1 phosphorylation at T826 upregulated repair of crosslinks, thus resulting in cisplatin resistance, the levels of crosslink DNA was measured using modified comet assay. In brief, the experiment was performed according to the manufacture's instruction of CometAssay Kit (Trevigen) where the modified comet assay was performed in cells treated with 10 μM cisplatin for 2 hours and released into cisplatin free medium at indicated times followed by 20 Gy of ionizing radiation (IR) to introduce random DNA double strand breaks before harvesting. For transfections, cells were transfected with siRNA 24 hours before plasmid transfection for another 24 hours. Cells were then subjected to modified comet assay.
As shown in
Although ATR inhibition resulted in elevated sensitivity to cisplatin in OC cells, the unpredictable side effects generated by variety of ATR-related pathways are undesirable for chemotherapy. As such, it was determined whether And-1 inhibition could overcome cisplatin resistance in OC cells. To this end, And-1 depletion by siRNAs significantly increased the cell sensitivity to cisplatin in both IGROV1 CR and OV90 CR cells (
Clonogenic survival assays were also performed. In brief, cells were seeded in 6-well dishes at a density of 500 cells per dish then treated with the cisplatin at the indicated concentrations (See
Analyses using data from The Cancer Genome Atlas (TCGA) indicated that expression levels of And-1/WDHD1 were significantly upregulated in most of the tested cancers but not normal tissues (
To evaluate the potency of And-1 inhibitors in vivo, IGROV1 or MCF7 cells were subcutaneously implanted into nude mice to form ovarian or breast tumors, respectively. In brief, 5-6 week-old female BALB/c athymic nude mice (CByJ.Cg-Foxn1nu/J, weighting 20-25 grams) were inoculated subcutaneously by injecting the OC cells, cisplatin resistant OC cells or breast cancer cells suspended in 100 μl ice-cold Matrigel/PBS (1:1, V:V; 5×106/mouse) into the dorsal flank of each mouse. When the average tumor volume reached 100-150 mm3, the mice were randomized into subsequent experiment groups. For tumors treated with CH3 only, CH3 was given at 0 mg/kg, 20 mg/kg and 40 mg/kg via intraperitoneal injection twice a week for three weeks. For combinational treatment, cisplatin, CH3, and BZA were then given intraperitoneally at 8 mg/kg, 20 mg/kg and 2 mg/kg respectively twice a week for three weeks. The relative tumor volumes were calculated using the formula: a×(b2)/2, for which a and b represented the longest and shortest diameters, respectively. During the drug treatment, the tumor volume and body weight were measured. Tumor samples were collected 4 days after final administration of the drug.
As shown in
To determine whether And-1 inhibitors can overcome cisplatin resistance in OC cells, the synergy of And-1 inhibitors and cisplatin in IGROV1 CR cells was examined. As shown in
To assess the efficacy of combination therapy with And-1 inhibitor and cisplatin in vivo, an IGROV1 CR xenograft was treated with vehicle, And-1 inhibitors, cisplatin or combination of cisplatin and And-1 inhibitor via intraperitoneal injection. Similar to cell-based assay result, it was found that the combinational treatments of CH3 and cisplatin, or BZA and cisplatin had excellent synergy in reducing tumor growth as compared to cisplatin treatment alone (
IHC analysis of tumor samples showed that CH3 and BZA suppressed expression of And-1, indicating that CH3 and BZA indeed targeted And-1 in vivo (
The liquid chromatography conditions of reference substance BF-175:
The chromatograph is Essentia LC-16 high performance liquid chromatograph, Dionex UltiMate 3000 liquid chromatograph; chromatographic column is XAqua Ph 5 μM 100 A 4.6×250 mm; column temperature is 35° C.; mobile phase is acetonitrile/water 70:30; flow rate is 1.000 mL/min; injection volume 10 μL; detection wavelength 315 nm. Pipette 20 μL of 1 mg/mL acetonitrile stock solution into 4 10 mL volumetric flasks, add 4 mL 0.1M hydrochloric acid (or 4 mL 0.1M sodium hydroxide) to each, keep in a 37° C. water bath, respectively take out at 5 min, 1 h, and dilute with acetonitrile To the mark, mix well, filter, and inject. The stability of the sample in 0.1M hydrochloric acid and sodium hydroxide was investigated.
Compound Liquid Chromatography Conditions:
The chromatograph is Essentia LC-16 high performance liquid chromatograph, Dionex UltiMate 3000 liquid chromatograph; chromatographic column is XAqua Ph 5 μM 100 A 4.6×250 mm; column temperature is 35° C.; mobile phase is acetonitrile/water 60:40; flow rate is 1.000 mL/min; injection volume 10 μL; detection wavelength 313 nm. Pipette 200 μL of 0.1 mg/mL acetonitrile stock solution into 8 10 mL volumetric flasks, add 4 mL 0.1M hydrochloric acid (or 4 mL 0.1M sodium hydroxide) to each, keep in a 37° C. water bath, and take them out at 5 min, 2 h, 5 h, 19 h respectively. Dilute to volume with acetonitrile, mix well, filter, and inject. The stability of the sample in 0.1M hydrochloric acid and 0.1M sodium hydroxide was investigated.
The results are detailed in
The above results prove that BF-175 is extremely unstable under acidic conditions, while its derivatives are stable under the same conditions.
Example 15. RT-PCR Detection Assay
-
- 1. Experimental reagent
- 2. The experimental steps are as follows:
- 2.1 AN3CA cell recovery and culture (10% serum 1% double antibody in MEM medium);
- 2.2 passages (passage at a ratio of 1:2);
- 2.3 Place 6 well plates when the cell status is good, 5×105Well, culture overnight;
- 2.4 adding medicine: diluting the final concentration of the drug with a culture medium to 10 μM, 2 mL/well;
- 2.5 adding medicine for 48 h, and then extracting RNA;
- 2.5.1 removing the culture medium, adding 0.5 mL/hole Trizol to lyse the cells, repeatedly blowing and beating the cells by a pipette after 5 min, completely lysing the cells, transferring the cells into a 1.5 mL EP tube without RNase, and standing the tube for 5 min;
- 2.5.2 adding 200 mul chloroform, shaking rapidly, mixing, standing for 10 min 12000 rpm/15 min;
- 2.5.3 carefully taking out the supernatant, transferring the supernatant into an EP tube, adding isopropanol with the same volume, reversing, uniformly mixing, standing for 10 min, and centrifuging at 12000 rpm/10 min;
- 2.5.4 removing the supernatant, adding 1 mL of 75% ethanol for washing, centrifuging at 8000 rpm/5 min once, carefully removing the supernatant, standing at room temperature for 10 min to volatilize the ethanol, and dissolving the precipitate with 40 μL of DEPC treated water when the precipitate becomes transparent;
- 2.5.5 RNA concentration was measured and DEPC-treated water was diluted to 100 ng/μL
- 2.6 reverse transcription: Kit Takara (PrimeScript RT reagent Kit (Perfect Real Time)
RT-PCR results show that all the compounds except compounds VI and VII can reduce the relative mRNA expression level of the SREBP target gene SCD-1 to different degrees, wherein the activities of compounds II and IV are better than those of BF-175.
Although benzoxaborole and travaborole also have the structure of benzo borone hetero-oxolane (structural formula shown in
-
- 1. The experimental steps are as follows:
- 1.1 AN3CA cell recovery and culture (MEM medium, 10% serum, 1% double antibody);
- 1.2 Passages (1:2 ratio passage);
- 1.3 When the cell state is good, laying 6-hole plates, 500/hole, and culturing overnight;
- 1.4 Adding medicine: diluting the final concentration of the drug with a culture medium to 10 μM, 2 mL/well;
- 1.5 Adding medicine in a liquid changing mode every two days;
- 1.6 Frequent observation. The culture was terminated when macroscopic colonies appeared in the culture dish. The supernatant was discarded, carefully rinsed 2 times with PBS and air dried. Cells were fixed with 5 mL of 4% paraformaldehyde for 30 minutes or with methanol for 10 minutes. And (3) removing the stationary liquid after air drying, adding a proper amount of GIMSA, dyeing for 10-30 minutes by using the dyeing liquid, then slowly washing away the dyeing liquid by using running water, and air drying.
- 1.7 Plates were inverted and overlaid with a piece of transparent film with grid and clones were counted either directly with the naked eye or with a microscope (low power) for clones larger than 10 cells. And finally calculating the clone formation rate.
- 1.8 Percent colony formation rate (number of clones/number of inoculated cells)×100%
The results of the clonogenic experiments are shown in
-
- 1. The experimental steps are as follows:
- 1.1 AN3CA cell recovery and culture (MEM medium, 10% serum, 1% double antibody);
- 1.2 Passages (passage at a ratio of 1:2);
- 1.3 Place 96-well plate when the cell status is good, 1×104 Well, culture overnight;
- culture medium with 10% defatted serum
- 1.4 Adding medicine: diluting the final concentration of the drug with a culture medium to 10 μM, and treating for 48 h at 100 μL/well;
- culture medium with 10% defatted serum
- 1.5 Addition of 2 μL MTS/well, care taken to prevent bubble formation, 490 nm data after 1-4 h
The result is shown in
The results show that the compound has different degrees of inhibition effects on IGROV1 CR and OV90 CR ovarian cancer cells, and the representative compound III has IC50 for inhibiting And-1 in IGROV1 CR cells of 7.8 μM.
Example 19. Western Blot Detection of And-1 Protein Expression Experiment1. Experimental Steps:
Select the A549 cells in good growth condition and inoculate them in a 60 mm petri dish at a density of 500,000. After culturing in a constant temperature incubator at 37° C. and 5% CO2 for 24 hours, add different compounds of the same concentration (5 μM) and continue culturing for 24 hours before collecting Cells were subjected to Western blotting experiments to compare with untreated cells to detect the inhibitory effect of each compound on the expression of And-1 protein.
The results are shown in
Therefore, the compounds of Formula I can inhibit the activities of Srebp and And-1 in various cell lines to varying degrees. Among them, the representative compounds III, X, and II have a high inhibitory effect on the Srebp of human endometrial cancer AN3CA cells at 10 μM, and have a good effect on the And-1 protein of human ovarian cancer cells IGROV1, OV90 and lung cancer cell A549. The inhibitory activity. The compound represented by formula I is expected to be an antitumor drug or a radiochemotherapy sensitizer.
Claims
1. A compound represented by formula I or a pharmaceutically acceptable salt thereof:
- wherein, R1 is independently mono-or poly-substituted with hydrogen, trifluoromethyl, trifluoromethoxy, nitro, cyano, halogen, C1-C10 alkyl, or —OR2,
- wherein R2 is independently selected from hydrogen or C1-C10 alkyl.
2. The compound of claim 1, wherein the at least one compound represented by formula I or a pharmaceutically acceptable salt thereof comprises a compound selected from the group consisting of
3. The compound of claim 1, wherein the compound represented by formula I or a pharmaceutically acceptable salt thereof comprises a compound represented by formula III,
4. A method for treating a subject in need thereof, the method comprising: administering to a subject having one or more tumors a therapeutically effective amount of a pharmaceutical composition comprising the compound represented by formula III, and at least one pharmaceutically acceptable excipient, wherein, following administration, anti-tumor activity is increased in the subject.
5. The method of claim 4, wherein the one or more tumors comprises a testicular tumor, ovarian tumor, cervical tumor, kidney tumor, bladder tumor, head-and-neck tumor, liver tumor, stomach tumor, lung tumor, endometrial tumor, esophageal tumor, breast tumor, cervical tumor, central nervous system tumor, germ cell tumor, prostate tumor, neuroblastoma, sarcoma, melanoma, mesothelioma, osteogenic sarcoma, or any combination thereof.
6. The method of claim 4 wherein the one or more tumors comprises a breast tumor, an ovarian tumor, or any combination thereof.
7. The method of claim 4, wherein the subject is resistant to at least one anticancer drug therapy.
8. The method of claim 7, wherein the at least one anticancer drug therapy comprises one or more platinum-based chemotherapeutics.
9. A method of preparing at least one compound of claim 1,
- the method comprising: a. reacting a compound represented by Compound A with concentrated hydrochloric acid and paraformaldehyde to obtain a compound represented by Compound B; b. reacting a compound represented by Compound B with triphenylphosphonium in an aprotic solvent to obtain a compound represented by Compound C; c. producing a compound represented by Compound E from compounds represented by Compound C and Compound D under strong base conditions; d. reacting a compound represented by Compound E with trifluoromethanesulfonic anhydride in dichloromethane to obtain a compound represented by Compound F; e. reacting a compound represented by Compound F with pinacol diboronic acid or diboronic acid under palladium salt catalyst to obtain a compound represented by Compound G; and f. reacting a compound represented by Compound G with a reducing agent such and adding acid to obtain a compound represented by Compound H, wherein the reducing agent comprises sodium borohydride, tetrahydroaluminum lithium, or any combination thereof;
- wherein:
- wherein R1 is independently mono-or poly-substituted with hydrogen, trifluoromethyl, trifluoromethoxy, nitro, cyano, halogen, C1-C10 alkyl, or —OR2,
- wherein R2 is independently selected from hydrogen or C1-C10 alkyl.
10. The method of claim 9, wherein the strong base conditions of step (iii) are strong alkaline conditions comprising sodium methoxide, sodium ethoxide, sodium tert-butoxide, sodium isopropoxide, butyl lithium, sodium hydroxide, lithium hydroxide, potassium hydroxide, or any combination thereof.
11. The method of claim 9, wherein steps (iii) and (v) are performed at about 80° C.
12. The method of claim 9, wherein steps (iv) and (vi) are performed at about 0° C.
13. The method of claim 9, wherein the palladium salt catalyst of step (v) comprises palladium acetate, palladium chloride, tetrakis(triphenylphosphorus) palladium, bis(acetonitrile) palladium dichloride, bis(triphenylphosphorus) palladium chloride, 1,1′-[bis(diphenylphosphorus)ferrocene]palladium dichloride, bis(benzonitrile)palladium dichloride, 1,1′-[bis(di-tert-butylphosphorus)ferrocene] Palladium dichloride, bis(tricyclohexylphosphorus) palladium dichloride, bis(o-toluene)palladium dichloride, or any combination thereof.
14. The method of claim 9, wherein step (v) is performed under an alkaline condition, wherein the alkaline condition comprises a base comprising sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, sodium fluoride, potassium fluoride, or any combination thereof.
15. The method of claim 9, wherein step (v) is performed in a polar solvent, wherein the polar solvent comprises N, N-dimethylformamide, dimethylsulfoxide, acetonitrile, acetone, methyl ethyl ketone, 1,4-dioxane, water, or any combination thereof.
16. The method of claim 4, further comprising administering to the subject one or more anticancer drugs or one or more platinum-based chemotherapeutics.
17. The method of claim 16, wherein the one or more anticancer drugs comprises gemcitabine, methotrexate, vinblastine, adriamycin, or any combination thereof, and wherein the one or more platinum-based chemotherapeutics comprises cisplatin, carboplatin, nedaplatin, satraplatin, picoplatin, phenanthriplatin, triplatin tetranitrate, or any combination thereof.
18. The method of claim 4, wherein the anti-tumor activity comprises reducing cell proliferation, reducing tumor growth, reducing tumor volume, reducing tumor burden, reducing tumor load, reducing the number of metastatic lesions, or any combination thereof.
20180230169 | August 16, 2018 | Ramanujachary et al. |
111333672 | June 2020 | CN |
WO 2019140265 | July 2019 | WO |
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Type: Grant
Filed: Sep 14, 2022
Date of Patent: Mar 25, 2025
Patent Publication Number: 20230129381
Assignee: The George Washington University (Washington, DC)
Inventors: Wenge Zhu (Germantown, MD), Jing Li (Arlington, VA), Yiliang Li (Tianjin)
Primary Examiner: Savitha M Rao
Application Number: 17/944,506